title: Protein phosphatase activity in human keratinocytes cultured from normal epidermis and epidermis from patients with harlequin ichthyosis.
abstract: We investigated serine/threonine protein phosphatase (PP) activity and the expression of PP2A during growth and differentiation of epidermal keratinocytes in culture. Keratinocyte PP activity was strongly inhibited by calyculin A and okadaic acid, indicating that the activity was mainly due to PP2A and PP1. The phosphatase activity decreased to about 20% of the initial (day 1) level by the time of confluence and to about 10% at day 7 postconfluence. In contrast to activity, the level of expression of the PP2A catalytic subunit protein and the mRNA for the two isoforms increased slightly over the period of growth. Keratinocyte differentiation was shown by a significant increase in profilaggrin expression after confluence. Keratinocytes were also cultured from individuals affected with harlequin ichthyosis. This severe hyperkeratotic skin disorder has abnormal lipid structures and is blocked in the PP2A-dependent conversion of phosphorylated profilggrin to the non-phosphorylated filaggrin. The PP activity in harlequin cultures was lower than in normal cultures (about 20% of the subconfluent normal control value) and decreased even further in confluent cultures. In contrast, the level of expression of the PP2A catalytic subunit protein and mRNA for the two isoforms was similar to that of normal keratinocytes and increased with confluence. These results suggest that PP activity in keratinocytes is regulated in a post-translational manner; they also support the possibility of impaired or reduced function of PPs in harlequin ichthyosis.
authors.affiliation: Department of Oral Biology, University of Washington, Seattle 98195-7132, USA.
authors.forename: E
authors.initials: E
authors.lastname: Kam
authors.forename: W
authors.initials: W
authors.lastname: Nirunsuksiri
authors.forename: B
authors.initials: B
authors.lastname: Hager
authors.forename: P
authors.initials: P
authors.lastname: Fleckman
authors.forename: B A
authors.initials: BA
authors.lastname: Dale
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D018929:Cell Culture Techniques; D004817:Epidermis / Q000201:enzymology*; D000091344:Filaggrin Proteins; D006801:Humans; D007057:Ichthyosis / Q000201:enzymology* / Q000473:pathology; D015603:Keratinocytes / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*
chemical_list: C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D010749:Phosphoprotein Phosphatases